This invention relates to the preparation of peptides with C-terminal proline amide.
Many peptide hormones of relatively small molecular size carry an .alpha.-amide substituent at the C-terminus of the peptide chain, and this amide group is believed generally to play an important role in their biological activity. It is thought that these C-terminal amide groups are formed enzymatically after biosynthesis of the peptide chain -- A. F. Bradbury et al, Nature, 298, 686 (1982). Although some proteins and peptides can now be produced by genetic engineering techniques, amidation of the C-terminal amino acid similarly has to be performed as a separate step after the synthesis of the peptide chain. There is, accordingly, a need for effective amidation techniques which can be used to produce such peptides with an amide group at the C-terminus.
Synthetic chemical means of amidation tend to be unsatisfactory for this purpose because they are generally insufficiently selective, giving rise to unwanted side reactions. There may also be a loss in the physiological activity of the peptide under such reaction conditions. Enzymatic techniques for the amidation are therefore to be preferred.
One such enzymatic technique uses porcine pituitary to convert peptides which terminate in glycine to the corresponding des-glycine peptide amides -- A. F. Bradbury et al, Nature, 298, 686 (1982). Unlike the chemical amidation techniques, this enzymatic process is apparently selective and not accompanied by side reactions. On the other hand, the use of porcine pituitary does present several disadvantages, because the enzyme source is not readily available in commercial quantities, because it is difficult to obtain the enzyme in a sufficiently pure form, and because the enzyme is somewhat unstable. It should also be noted that this enzyme apparently has a requirement for C-terminal glycine in the substrate peptide.
Another enzyme which has been investigated for C-terminal amide formation in peptides is carboxypeptidase Y. This enzyme, which can be obtained from baker's yeast, is an exopeptidase and cleaves the C-terminal peptide bond to release successive amino acids from the C-terminus of the peptide chain. However, in common with other peptidases, this enzyme can also be used to catalyse the formation of peptide bonds -- Kazuyuki Morihara, Protein-Nucleic Acid-Enzyme, 26, 1979 (1981). K. Breddam et al in Carlsberg Res. Commun. 46, 121 (1981) describe the use of carboxypeptidase Y to catalyse the exchange of C-terminal amino acid residues in peptides for various other groups, including the conversion of peptides to peptide amides. This reference states that the yield of the reaction is strongly dependent on the nature of the C-terminal amino acid residue.
The best yields are obtained with peptide substrates in which a hydrophilic amino acid (e.g. glycine) serves as the leaving group, and the lowest yields are obtained with substrates in which a hydrophobic amino acid (e.g. valine or phenylalanine) serves as the leaving group. It also states that the penultimate amino acid residue of the substrate exerts a pronounced influence on the yield; and there is no disclosure of any reaction using a substrate in which the penultimate amino acid is proline. This follows a general belief in the art that peptides having proline as the penultimate C-terminal amino acid residue are resistant to such reactions.